Temperature and intrinsic Ca ²⁺ reshape TRPM4 pharmacology

Proteins operate in dynamic environments where ions, lipids, and temperature collectively define their properties, yet most studies rely on simplified conditions that overlook these intrinsic variables. Here, we show two such factors-temperature and Ca ²⁺ -profoundly remodel the function and pharmacology of TRPM4, an ion channel implicated in cardiac conduction, immune regulation, cancer, and intestinal fluid homeostasis. At physiological temperature and Ca ²⁺ , TPPO-previously considered a selective TRPM5 inhibitor inactive toward TRPM4-potently activates TRPM4, revealing strong synergy among temperature, Ca ²⁺ , and ligand binding. In contrast, Necrocide-1, a necroptotic activator targeting the same binding pocket, defies this logic: it opens TRPM4 without Ca ²⁺ but is antagonized by Ca ²⁺ . Meanwhile, the inhibitors NBA and CBA engage a nearby pocket, locking the channel in a non-conductive pre-open state. Our findings highlight that even rigid binding pockets can exhibit temperature-dependent ligand recognition, revealing hidden pharmacology and informing selective, environment-aware therapeutic strategies.